A murine model of obesity implicates the association of inadequate levels of a serine protease, designated adipsin, with obesity in mice. This protein is synthesized in and secreted from adipocytes, and synthesis of mRNA encoding this protein appeared specific to adipocytes (Zusalak, K. M., et al. J Mol Cell Biol (1985) 5:419). The levels of adipsin mRNA and adipsin protein in adipose tissue, as well as the level of adipsin protein circulating in blood, are lowered in obese animals.
More specifically, Flier, J. S., et al. Science (1987) 237:405-408 showed that the abundance of adipsin mRNA in adipose tissue was increased during fasting in normal rats and in the form of diabetes due to streptozotocin-induced insulin deficiency, but was decreased in a hyperglycemic, hyperinsulinemic state accompanied by increased adipose mass induced by a continuous infusion of glucose, as well as in strains of genetically obese mice. Adipsin mRNA was also shown to be reduced when obesity was induced by injection of monosodium glutamate to newborn mice. However, adipsin expression levels did not change in a model of obesity obtained simply by overfeeding normal rats. The relationship of both adipsin mRNA levels in adipocytes and adipsin protein levels in serum to obesity in mice, and in humans is disclosed and described in PCT application publication no. W088/07681, incorporated herein by reference.
The gene encoding adipsin in the murine system has been cloned and sequenced. Cook, K. S., et al. Proc Natl Acad Sci USA (1985) 82:6480-6484 obtained the sequence for a cDNA clone from murine 3T3 differentiating adipocytes which hybridizes to an mRNA that is induced at least 100-fold during differentiation and that encodes a protein of 28 kd having substantial homology to various serine proteases such as trypsin, chymotrypsin and elastase. The protein appears to be produced, however, in glycosylated 37 kd and 44 kd forms. The genomic sequence encoding this protein has also been retrieved (Min, H. Y., et al. Nucleic Acids Research (1986) 14:8879-8891) and the protein has been shown to be secreted into the circulation by adipose tissue and sciatic nerve (Cook, K. S., et al. Science (1987) 237:402-404).
Despite the recovery of the coding sequences, the murine protein has not been produced recombinantly, nor has the protein been prepared in pure form.
The complement system is also known to involve serine proteases, and the complete amino acid sequence of the serine protease complement protein D has been determined from the isolated protein (Niemann, M. A., et al. Biochemistry (1984) 23:2482-2486). The complement system is a complex interacting array of 20 proteins which, in the main, is activated by antigen/antibody interactions, specifically by binding of the Cl protein to immunoglobulins of the IgG and IgM class through its Clq subcomponent (Bio Essays (1986) 4:249-253). The result of activation of the complement cascade appears related to destruction of invading microbial hosts, but also to inflammation and, in some cases, to disease states such as arthritis.
In addition to the immune system-activated portion of the complement cascade, there is an alternate pathway in which the serine protease, complement D, is a participant. In this system, the alpha-chain of C3 (120 kd) is hydrolyzed to obtain C3a (9 kd) and C3b which is assembled with protein B to obtain C3bB, which is, in turn, hydrolyzed through the catalysis of complement factor D to obtain C3bBb. C3bBb catalyzes the hydrolysis of C3 to continue the cycle. Both C3a and C3b may directly interact with adipocytes or other cells related to fat metabolism. In addition, C3b can be further manipulated to obtain additional factors such as C5a and Ba which are also thus interactive.
Although the murine adipsin gene has been obtained, and synthetic peptides designed from the deduced amino acid sequence have been successfully used to obtain antibodies immunoreactive with circulating adipsin in mice, the human gene has not been available, nor are the murine antibodies generated to the synthetic peptides cross-reactive with human adipsin. Thus, the insights obtained with respect to murine metabolism have not, prior to this invention, been accessible to human systems. Furthermore, the purified murine protein has not been obtained. The present invention provides a DNA encoding a human protein which has both the energy regulating activity of adipsin and the macrophage lysis stimulation activity of complement factor D. The invention also provides purified murine adipsin.
The nexus between adipsin activity and complement D activity has not been suggested, although Mole, J. E., et al., in an abstract presented at the 12th International Complement Workshop, held 18-21 Sep. 1987, reported a partial cDNA clone for complement D which, when subjected to a computerized search, revealed a possible evolutionary relationship to the sequence reported for mouse adipsin, i.e., the cDNA complementary to the adipsin mRNA is induced during adipocyte differentiation.
Because serine proteases in general display a high degree of homology, homology alone does not necessarily lead one to conclude that the activity in assays for adipsin and for complement D would be shared. The present invention establishes these dual activities for this protein.